T and B cell egress from lymphoid organs is necessary for immune surveillance and effector cell function. Work over the last 25 years has provided a detailed cellular and molecular picture for how cells enter tissues from the blood. By contrast, very little has been defined about how cells exit tissues and return to circulation. The clinical importance of lymphocyte egress and lymphocyte recirculation has been revealed by the finding that the immunosuppressive molecule, FTY720, acts by inhibiting egress from lymphoid organs. Work over the last five years has established a critical role for S1P receptor-1 (S1P1) in lymphocyte egress from lymphoid organs. S1P1 has also been found to be a control point for egress regulation. The lymphocyte activation marker, CD69, acts as a negative regulator of S1P1 and lymphocyte egress. However, the mechanism by which lymphocyte S1P1 promotes egress, and the mode of CD69 mediated regulation of S1P1 remain undefined. This proposal will address the cellular and molecular requirements for lymphocyte egress and mechanisms associated with egress regulation through three specific aims. The first Aim will use a combination of cellular and multiphoton imaging approaches to characterize the S1P1-dependent step in lymphocyte egress from lymph nodes. Aim 2 will focus on determining the mechanism of CD69 mediated inhibition of S1P1 function and lymphocyte egress. The final Aim will use genetic approaches to identify additional molecules required for lymphocyte egress. This aim will build upon a collaboration with Christopher Goodnow in Australia to identify and characterize mutant mouse lines that exhibit alterations in lymphocyte trafficking. As part of this effort we will fine map and characterize the locus responsible for the thymic egress defect in Cataract Shionogi (CTS) mice. The information obtained through these studies should point to new targets for the development of immunomodulatory molecules.